Basic insight on plant

@ Roxana Portieles, Camilo Ayra, Orlando Borrás Plant Division, Center for Genetic Engineering and Biotechnology, CIGB Ave 31 / 158 and 190, Playa, PO Box 6162, Havana 10 600, Cuba E-mail: [email protected] ABSTRACT REVIEW Defensins are small cysteine-rich peptides with antimicrobial activity. Plant defensins have a characteristic three- dimensional folding pattern that is stabilized by eight disulfide-linked cysteines. This pattern is very similar to defense peptides of mammals and insects, suggesting an ancient and conserved origin. Functionally, these proteins exhibit a diverse array of biological activities, although they all serve a common function as defenders of their hosts. Plant defensins are active against a broad range of phytopathogenic fungi and even against human pathogens, which indicates their potential in therapeutics. This review briefly describes the distribution, structure, function and putative modes of antimicrobial activity of plant defensins and reflects their potential in agriculture and medical applications. Key works: , cysteine-rich peptides, mode of action

Biotecnología Aplicada 2006;23:75-78 RESUMEN Conocimientos básicos acerca de las defensinas de plantas. Las defensinas son pequeños péptidos ricos en cisteína con actividad antimicrobiana. Las defensinas de plantas poseen un característico patrón de plegamiento tri-dimensional estabilizado por ocho puentes disulfuro de residuo de cisteinas. Este patrón comparte una alta semejanza con los péptidos de defensa de mamíferos e insectos, sugiriendo un origen común conservado. Funcionalmente estas proteínas exhiben un conjunto diverso de actividades biológicas, aunque todas están al servicio de una función común, como defensoras de sus hospederos. Las defensinas de plantas se activan contra un amplio rango de hongos fitopatogénicos e incluso contra patógenos humanos, lo cual indica su potencial para el desarrollo de terapéuticos. El objetivo de esta revisión es describir brevemente la distribución, función y los modos de actividad antimicrobiana propuestos para las defensinas de plantas, así como reflejar las aplicaciones de las mismas en la agricultura y la medicina. Palabras claves: defensinas, péptidos ricos en cisteína, modo de acción

Introduction Plants are constantly exposed to environmental stres- cysteine-rich peptides ranging from 45 ses and the attacks of pathogenic organisms. Survival to 54 amino acids, and are positively charged. The 1. Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner HY, Hunt MD. Sys- under these conditions demands quick defense first plant defensins were isolated from (Tri- temic acquired resistance. Plant Cell 1996; responses to inhibit pathogen spread after initial ticum aestivum) and (Hordeum vulgare), and 8:1809-19. infection and thus to limit disease. Among these defense were initially classified as a subgroup of the 2. Kawata M, Nakajima T, Yamamoto T, responses are a rapid oxidative burst, local transcrip- family called the g- since they showed a si- Mori K, Oikawa T, Fukumoto F, Kuroda S. tional activation of defense-related genes, and the milar size and the same number of disulfide bridges as Genetic Engineering for Disease Resistance in Rice (Oryzasativa L.) using antimicrobial generation of yet unknown systemic signals that trigger a and b-thionins [3]. Subsequent identification of Peptides. JARQ 2003;37:71-6. and induce a state of systemic immunity [1]. Small other g-thionin-like proteins in other plant families, 3. Colilla FJ, Rocher R, Mendez E. g-Pu- play an important role as part together with structural information, revealed striking rothionins: amino acid sequence of two of the plants` natural defense system against infectious differences between g-thionins and classical thionins polypeptides of a new family of thionins from wheat endosperm. FEBS Lett 1990; microorganisms, by recognizing a broad range of mi- [4]. This class of proteins was later renamed as “plant 270:191-4. crobes. Hundreds of antifungal peptides and proteins defensins” [5], due to structural and functional simi- are known, with more being discovered almost daily. 4. Terras FRG, Eggermont K, Kovaleva V, larities with insect and mammalian defensins [4]. To Raikhel NV, Osborn RW, Kester A, Ress SB, Genetic engineering has provided a new strategy date, plant defensins have been isolated from seeds Torrekens S, Van Leuven F, Vanderleyden for improving disease resistance through cellular and of various monocot and dicot species or identified by J. Small cysteine-rich antifungal proteins from radish: their role in host defense. Plant molecular tools [2]. Based on in vitro analysis, many the sequencing of cDNA clones. Cell 1995;7:573-88. antimicrobial peptides have inhibited the growth of The plant defensin family is quite diverse regarding 5. Broekaert WF, Terras FRG, Cammue a broad range of plant pathogenic fungi and some amino acid composition as only the eight structure- BPA, Osborn RW. Plant defensins: novel bacteria. The enhancement of disease resistance in stabilizing cysteines appear to be conserved among antimicrobial peptides as components of crops has contributed significantly to increase their the host defense system. Plant Physiol all plant defensins [6]. The variation in the primary 1995;108:1353-8. productivity and decrease the application of sequences may account for the different biological pesticides, which can affect human health and the activities reported for plant defensins. The three- 6. Thomma BPHJ, Thevissen K, Cammue BP. Plants defensins. Planta 2002;216: environment. dimensional structure of plant defensins comprises a 193-202. triple-stranded b-sheet with an a-helix in parallel, Plant defensins 7. Almeida MS, Cabral KM, Kurtenbach E, stabilized by four disulfide bridges [7-8]. This Almeida FC, Valente APJX. Solution structure The main groups of antimicrobial peptides found structure resembles that of insect and mammalian of Pisum sativum defensin 1 by high resolution NMR: plant defensins, identical in plants are thionins, defensins and lipid transfer defensins except that insect defensins lack the domain backbone with different me-chanisms of proteins. Plant defensins are small (c.a. 5kDa), basic, corresponding to the amino-terminal b-strand of plant action. Mol Biol 2002;315:749-57.

@ Corresponding author Roxana Portieles et al. Basic insight about plant defensins

defensins (Figure 1). Mammalian defensins, on the A B C other hand, not only have a triple-stranded, antiparallel b-sheet, which is roughly comparable in size and spatial orientation to that occurring in plant defensins, but also the a-helix appears to be approximately in the same position relative to the b-sheet [9-10]. The fact that plant defensins, belong to a superfamily of similarly folded antimicrobial peptides that has representati-ves in vertebrates, invertebrates, and plants, suggests that these defense molecules predate the evolutionary divergence of animals and plants. tructure-activity relationships Figure 1. Three-dimensional structure of some known defensins isolated from different organisms. Structures S were downloaded from the protein data bank, PDB accession ID numbers: A. Human b defensin HBD-1 and modes of action (PDB 1E4S) (ref 9). B. Mouse defensin MBD-8 (PDB 1E4R) (ref 9). C. Plant Defensin (PDB 1TI5) (ref 10). The structure-activity relationships and modes of action for most of the plant defensins remain unknown. cation-resistant permeabilization correlates with the Not all plant defensins have the same mode of action. inhibition of fungal growth. Reduction of the antimi- Some of them exhibit potent antifungal activity in crobial activity in the presence of divalent cations, vitro at micromolar concentrations against a broad especially Ca2+, has also been observed for insect and spectrum of filamentous fungi. Morphogenic anti- mammalian defensins [18]. A partial explanation for 8. Fant F, Vranken WF, Borremans FAM. fungal defensins reduce hyphal elongation and induce this effect could be that an increase in ionic strength The three-dimensional solution structure of Aesculus hippocastanum antimicro- hyperbranching, whereas non-morphogenic defensins weakens the electrostatic charge interactions required bial protein 1 determined by 1H nuclear reduce hyphal elongation without causing any mor- for the initial interaction between the peptides and magnetic resonance. Proteins 1999;37: phological distortions [5-11-12]. The plant defensins the membrane. 388-403. Rs-AFP1 and Rs-AFP2 from radish (Raphanus sa- For most plant defensins, molecular components 9. Bauer F, Schweimer K, Kluver E, Cone- tivus), and alfAFP isolated from seeds of the Medicago involved in signaling and putative intracellular targets jo JR, Forssmann WG, Rosch P, Adermann K, Sticht H. Structure determination of sativa (alfalfa) plants [13], are examples of potent an- remain unknown. At present there are two tentative human and murine beta-defensins reveals tifungal proteins, causing morphological distortions models for the mode of action of plant defensins in structural conservation in the absence of significant sequence similarity. Protein Sci of the fungal hyphae, resulting in hyperbranched fungal relation to sphingolipids, that are one of the three major 2001;10:2470-9. structures. The antifungal activity of plant defensins, types of lipids found in eukaryotic membranes, along whether morphogenic or not, is reduced by increasing with sterols and phosphoglycerolipids. They associate 10. Liu YJ, Cheng CS, Liu YN, Hsu MP, Chen CS, Lyu PC. Solution structure of plant the ionic strength of the fungal growth assay medium. with sterols in the plasma membrane to form patches defensin (unpublished). However, the antagonistic effect of ions is strongly or rafts that are highly enriched in glycosyl- 11.Thomma BP, Cammue BP, Thevissen K. dependent on the fungus and thus on the conformation phosphatidyl-inositol (GPI)-anchored membrane Mode of action of plant defensins suggests of the putative target site. Ionic strength antagonism proteins [19]. The interaction facilitates the insertion therapeutic potential. Curr Drug Targets was found to be due to cations, with divalent cations of these peptides in the plasma and leads to fungal Infect Disord 2003;3:1-8. being at least one order of magnitude more potent growth arrest. Alternatively, GPI-anchored proteins 12.Spelbrink RG, Nejmi AA, Thomas JS, than monovalent cations. In general, the antifungal could act as docking sites for these defensins and Dilip MS, Hockerman GH. Differential Antifungal and Calcium Channel-Bloc- activity of plant defensins is slightly more reduced facilitate their insertion into the fungal plasma king Activity among Structurally Related by Ca 2+ than by Mg 2+ [14]. Alfalfa seed defensin, membrane, leading to alterations in membrane Plant Defensins. Plant Physiology 2004; MsDef1, strongly inhibits the growth of Fusarium permeability. Likewise, mammalian GPI-anchored 135:2055-67. graminearum in vitro, and its antifungal activity is proteins have been shown to act as receptors for a 13.Gao AG, Hakimi SM, Mittanck CA, 2+ Wu Y, Woerner BM, Stark DM, Shah DM, markedly reduced in the presence of Ca . This anta- bacterial toxin, aerolysin, that causes pore formation Shah DM, Liang J, Rommens CM. Fungal gonistic effect of cations was also observed in the case [20]. Recently, the theory is gaining support in that pathogen protection in by ex- of plant defensin-induced membrane permeabilization many cationic peptides exert their anti-microbial pression of a plant defensin peptide. Nat Biotechnol 2000;18:1307-10. through specific interaction with high-affinity binding activity not only through the permeabilization of the sites on fungal cells but no ion-permeable pores are membrane, but also through cytoplasmic targets [21]. 14.Osborn RW, De Samblanx GW, The- vissen K, Goderis I, Torrekens S, Van Leuven formed in artificial lipid bilayers, nor are there changes Plant defensins are active against a broad range of F, Attenborough S, Ress SB, Broekaert WF. in their electrical properties [15-16]. phytopathogenic fungi and even against human pa- Isolation and characterisation of plant defensins from seeds of Asteraceae, Fa- Antifungal plant defensin, Rs-AFP2, appears to act thogens. Certain members within the plant defensin baceae, Hippocastanaceae and Saxifra- primarily at the cell membrane [17] and induces rapid family also display other biological activities, including gaceae. FEBS Lett 1995;368:257-62. Ca2+ uptake and K+ efflux from Neurospora crassa proteinase [22-23] and á-amylase [24] inhibitory 15.Thevissen K, Osborn RW, Acland DP, hyphae and it may thus inhibit the growth of filamentous activities and the inhibition of protein translation [3], Broekaert WF. Specific, high affinity fungi by disrupting cytosolic Ca2+ gradients essential which may contribute to their role in defense. binding sites for an antifungal plant defensin on Neurospora crassa hyphae for hyphal tip growth. Thesivven and colleagues [16- and microsomal membranes. J Biol Chem 17] have suggested that defensin initiates this response Applications 1997;272:32176-81. by its interaccion with a membrane-bound receptor To date, several types of antimicrobial peptides have 16.Thevissen K, Osborn RW, Acland DP, rather than by permeabilizing the membrane by the been isolated and characterized and the applications Broekaert WF. Specific binding sites for direct defensin-lipid interaction. of plant defensins are diverse. They can be used as re- an antifungal plant defensin from dahlia (Dahlia merckii) on fungal cells are Membrane permeabilization induced by plant de- sistance traits in transgenic plants, resulting in an required for antifungal activity. Mol Plant fensins in Neurospora crassa is biphasic, depending enhanced protection against pathogen attack (Figure Microbe Interact 2000;13:54-61. on the plant defensin dose. At high defensin levels (10 2). The constitutive expression of a novel alfalfa 17.Hevissen K, Ghazi A, De Samblanx GW, to 40 mM), strong permeabilization is detected that defensin gene in potato provides high levels of field Brownlee C, Osborn RW, Broekaert WF. Fungal membrane responses induced by can be suppressed by cations in the medium. At lower resistance against dahliae, the causal agent of the plant defensins and thionins. J Biol Chem defensin levels (0.1 to 1 mM), a weaker, but more agrono-mically important “early dying” disease of 1996;271:15018-25.

76 Biotecnología Aplicada 2006; Vol.23, No.2 Roxana Portieles et al. Basic insight about plant defensins

of the fungal disease process and by the application 18.De Samblanx GW, Goderis IJ, The- of multiple plant defensins (a “cocktail” of defensins) vissen K, Raemaekers R, Fant F, Borremans for a synergistic effectiveness at the right place and F, Acland D P, Osborn R W, Patel S, Broe- kaert WF. Mutational analysis of a plant time. Since plant defensins are protein based, it is defensin from Radish (Raphanus sativus presently not feasible to synthesize large amounts of L.) reveals two adjacent sites important for antifungal activity. J Biol Chem 1997; plant defensins as in the large-scale syntheses of 272:1171-9. chemical fungicides in a factory. One stumbling block 19.Bagnat M, Keranen S, Shevchenko A, preventing the application of plant defensins is the Simons K. Lipid rafts function in biosynthe- lack of an efficient way of producing large amounts of tic delivery of proteins to the cell surface in these natural antifungal proteins for commercial use. yeast. Proc Natl Acad Sci (USA) 2000;97: 3254-9. On the other hand, the control of eukaryotic pathogens constitutes one of the most challenging 20.Abrami L, Velluz MC, Hong Y, Ohishi K, Mehlert A, Ferguson M, Kinoshita T, Gisou problems in medicine. It is widely accepted among van der Goot F. The glycan core of GPI- clinicians, medical researchers, microbiologists and anchored proteins modulates aerolysin pharmacologist, that resistance will, in the binding but is not sufficient: the poly- peptide moiety is required for the toxin- very near future, leave healthcare professionals receptor interaction. FEBS lett 2002;512: without effective therapies for bacterial infections. 249-54. Fig 2. A cartoon showing how a plant has acquire a pathogen resistance state after spraying defensins in it while not treated As an example, it is now estimated that about half of 21.Xiong YQ, Yeaman MR, Bayer AS. In plants died. all Staphylococcus aureus strains found in many vitro antibacterial activities of platelet microbicidal protein and neutrophil de- medical institutions are resistant to such fensin against Staphylococcus aureus are potato. This protein also inhibits the growth of other as methicillin [29]. The emergence among enterococci influenced by antibiotics differing in of resistance to another useful and widely effective mechanism of action. Antimicrob Agents plant pathogens such as Alternaria solani and Fusarium Chemother 1999;43:1111-7. culmorum, the causal agents of potato early blight antibiotic, vancomycin [30], might accelerate the and wheat head scab, respectively [13]. On the other spread of vancomycin-resistant genes, via plasmids, 22.Wijaya R, Neumann GM, Condron R, Hughes AB, Polya GM. Defense proteins hand, plants that constitutively expressed a plant throughout other species, eventually limiting the from seed of Cassia fistula include a lipid defensin gene from B. oleracea and B. campestris were efficacy of this drug. Consequently, the priority for transfer protein homologue and a pro- tease inhibitory plant defensin. Plant Sci modified to substitute a single amino acid at each the next decades should be focused on the development 2000;159:243-55. position, and were individually introduced into rice of alternative drugs and/or the recovery of natural 23.Melo FR, Rigden DJ, Franco OL, Me- since rice and food production have experienced a molecules that would allow a consistent and llo LV, Ary MB, Crossi de Sa MF, Bloch C. critical damage from fungal rice blast caused by Mag- appropriate control of pathogen-caused diseases. Inhibition of by cowpea thionin: naporthe grisea, the main problem for crop yield and Ideally, these molecules should be as natural as possible, characterization, molecular modeling, and docking. Proteins Struct Funct Genet bacterial leaf blight caused by Xanthomonas oryzae, with a wide range of action over several pathogens, 2002;48:311-9. a serious rice disease in subtropical and tropical easy to produce, and not prone to induce resistance. 24.Zhang NY, Jones BL, Tao P. Purifica-tion countries. These two plant defensins from B. oleracea Antimicrobial peptides including defensins, and characterisation of a new class of insect and B. campestris conferred an effective resistance to isolated from a full range of organisms and species a-amylase inhibitors from barley. Cereal both rice blast and bacterial leaf blight, and the from bacteria to man, seem to fit this description. Chem 1997;74:119-22. modification of the defensin genes led to an increase They can be used as the leading molecules for the 25.Terras FR, Eggermont K, Kovaleva V, development of natural antibiotics, the basic element Raikhel NV, Osborn RW, Kester A, Ress SB, in the broad disease resistance spectrum in transge- Torrekens S, Van Leuven F, Vanderleyden nic rice [2]. of a novel generation of drugs for the treatment of J. Small cysteine-rich antifungal proteins The constitutive expression of plant defensins in bacterial and fungal infections [31-34]. In addition, from radish: their role in host defense. Plant Cell 1995;7:573-88. peripheral cells of the seeds in radish [25], the flower the wide spectrum of antimicrobial activities reported organs in tobacco [26], the leaves in pea [5], or the for these molecules suggests their potential benefit 26.Gu Q, Kawata EE, Morse MJ, Wu HM, Cheung AY. A flower-specific cDNA en- tubers in potato [27] is consistent with the first-line in the treatment of cancer [35] and viral [36-38] or coding a novel thionin in tobacco. Mol defense role of vulnerable tissues. Similarly, enhanced parasitic infections [39]. Gen Genet 1992;234:89-96. Different therapeutic applications of these com- resistance has been obtained in transgenic oil rapeseed, 27.Moreno M, Segura A, García-Olme- chinese cabbage and tomato plants by constitutive pounds, from topical administration to the systemic do F. Pseudothionin-St1, a potato peptide plant defensin expression [28]. These demonstrate treatment of infections, have been developed by active against potato pathogens. Eur J Biochem 1994;223:135-9. that plant defensins are important components of host several biotechnological companies [32]. Various defense. Moreover, they can be used to generate insect and mammalian defensins are currently being 28.Park HC, KangYH, Chun HJ, Koo JC, Cheong YH, Kim CY, Kim MC, Chung WS, transgenic crops with improved pathogen resistance. tested in clinical trials to examine their use in bacterial Kim JC, Yoo JH, Koo YD, Koo SC, Lim CO, The application of plant defensins to crops to and fungal infections. Micrologix biotech Inc. Lee SY, Cho M J. Characterization of a reduce losses caused by fungal pathogens provides developed different defensin variants which are stamen-specific cDNA encoding a novel plant defensin in Chinese cabbage. Plant two primary advantages over the conventional spray currently evaluated in a late-stage clinical trial to Mol Biol 2002;50:59-69. of chemical fungicides. First, plant defensins are prevent catheter-related infections caused by bacteria 29.Roder BL, Wandall DA, Frimodt-Mo- derived from plant seeds, roots and tubers, and are such as Pseudomonas aeruginosa and Staphylococcus ller N, Espersen F, Skinhoj P, Rosdahl VT. thus natural products. They have minimal harmful aureus, and for their potential to cure severe acne Clinical features of Staphylococcus aureus endocarditis: a 10-year experience in effects on human beings. Secondly, like any other and to prevent acute S. aureaus infections [40]. The Denmark. Archives Internal Medicine proteins, plant defensins quickly degrade to natural French company EntoMed is conducting preclinical 1999;159:462-9. studies with a defensin-like antifungal insect peptide elements. Essentially no “residues” are left after the 30.Novak R, Henriques B, Charpentier E, anti-fungal effectiveness expires. One drawback of to combat Candida albicans and Aspergillus Normark S, Tuomanen E. Emergente of plant defensins is their relatively lower potency fumigatus, which often cause fatal infections in vancomycin tolerante in Streptococcus pneumoniae. Nature 1999;399:590-1. compared to chemical fungicides. But the apparent immunocompromised patients [41]. Plant defensins, environmental safety of plant defensins can justify in contrast to insect and mammalian defensins, 31.De Lucca AJ. Antifungal peptides: potential candidates for the treatment of using them as alternative fungicides. This disadvantage interact with specific structures in the fungal fungal infections. Expert Opinion on can also be reverted by a more thorough understanding membrane, such as phosphorylinositol containing Investigational Drugs 2000;9:273-99.

77 Biotecnología Aplicada 2006; Vol.23, No.2 Roxana Portieles et al. Basic insight about plant defensins sphingolipids or glucosyl ceramides, which points approaches. The precise mode of action of plant towards a high selectivity and hence, interesting defensins is still unclear and most plant defensin perspectives for the treatment of fungal infections. molecular components involved in signaling and puta- Importantly, various plant defensins such a Dm- tive intracellular targets remain unknown. Not all plant AMP1, from Dahlia merckii seeds Hs-AFP1 from defensins act through the same mode of action. The fact Heuchera sanguinea seeds and Rs-AFP2 were found that some defensins have been found to interact with to be active against the human pathogen Candida fungal-specific components in the plasma membrane albicans at micromolar concentrations, which resulting in membrane permeabilization, makes them an indicates the potential of plant defensins for the attractive potential therapeutic source to treat fungal development of therapeutics. These make the infections and can be used as the leading molecules for antibiotic peptides a powerful mine of molecules that the development of antifungal medicines. On the other could become antimicrobial drugs in this new century. hand, among other diverse applications they can be used as resistance traits in transgenic crops, resulting in Concluding remarks enhanced protection against pathogen attack. Since genetic engineering using antimicrobial peptides is becoming a powerful tool for the introduction of new Acknowledgments traits in plants, many studies are in progress to improve The authors wish to thank to Mr. Yunior López Re- plant breeding for disease resistance by transgenic galón for the drawing.

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Received in october, 2005. Accepted for publication in december, 2005.

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